Polypeptides are susceptible to denaturation or enzymatic degradation in the blood, liver or kidney. Because of the low stability of polypeptides, it has been required to administer a polypeptide drugs in a sustained frequency to a subject in order to maintain an effective plasma concentration of the active substance. Moreover, since polypeptide drugs are usually administrated by infusion, frequent injection of polypeptide drugs causes considerable discomfort to a subject. Thus, there have been many studies to develop a polypeptide drug which has an increased circulating half-life in the blood, while maintaining a high pharmacological efficacy thereof. Such desirous polypeptide drugs should also meet the requirements of enhanced serum stability, high activity, applicability to various polypeptides and a low probability of inducing an undesired immune response when injected into a subject.
One of the most widely used methods for improving the stability of proteins is the chemical modification of a polypeptide with highly soluble macromolecules such as polyethylene glycol (“PEG”) which prevents the polypeptides from contacting with proteases. It is also well known that, when linked to a polypeptide drugs specifically or non-specifically, PEG increases the solubility of the polypeptide drug and prevents the hydrolysis thereof, thereby increasing the serum stability of the polypeptide drug without incurring any immune response due to its low antigenicity (Sada et al., J. Fermentation Bioengineering, 1991, 71: 137-139). However, such pegylated polypeptides have the disadvantages of lowering both the activity and production yield of an active substance as the molecular weight of PEG increases. An interferon conjugated with two activated PEGs as well as a PEG spacer which is linked to two polypeptides having different activities are disclosed in U.S. Pat. No. 5,738,846 and International Patent Publication No. WO92/16221, respectively; however, they do not show any distinctive effect in terms of prolonged activity of the physiologically active polypeptides in vivo.
It is also reported that the circulating half-life of a recombinant human granulocyte-colony stimulating factor (“G-CSF”) can be prolonged by covalently linking it to albumin through a hetero-bifunctional PEG (Kinstler et al., Pharmaceutical Research, 1995, 12(12): 1883-1888). However, the stability of the modified G-CSF-PEG-albumin is merely 4 times higher than that of authentic G-CSF and, thus, it has not yet been put to practical use.
As another approach for enhancing the in vivo stability of physiologically active polypeptides, an active polypeptide fused with a stable protein is produced in a transformant by using recombinant technologies. For example, albumin is known as one of the most effective proteins for enhancing the stability of polypeptides fused thereto and there are many such fusion proteins reported (International Patent Publication Nos. WO93/15199 and 93/15200, and European Patent Publication No. 413,622). However, a fusion protein coupled with albumin still has the problem of reduced activity.
U.S. Pat. No. 5,045,312 discloses a method for conjugating growth hormone to bovine serum albumin or mouse immunoglobulin using a cross-linking agent such as carbodiimide, glutaraldehyde, acid chloride, etc. in order to enhance the activity of the growth hormone. However, this method is solely aimed at enhancing the activity of a target growth hormone. In addition, the use of chemical compounds such as carbodiimide, glutaraldehyde, acid chloride, etc. as a cross-linking agent is disadvantageous due to their potent toxicity and non-specificity of reaction.
It has been reported that immunoglobulins are capable of acting as antibodies to exhibit antibody-dependent cell cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC), and sugar chains play an important role in the ADCC and CDC (Burton D., Molec. Immun. 22, 161-206, 1985). Notwithstanding the absence of sugar chains, an aglycosylated immunoglobulin has an blood half-life similar to that of the glycosylated one, however, its affinity to a complement or receptor decreases by 10 to 1000 folds to due to the deglycosylation (Waldmann H., Eur. J. Immunol. 23, 403-411, 1993; Morrison S., J. Immunol. 143, 2595-2601, 1989).
Although there have been many attempts to combine a physiologically active polypeptide with various macromolecules, all have failed to simultaneously increase the stability and the activity.
As an improved method for enhancing the stability of an active polypeptide and simultaneously maintaining the in vivo activity thereof, the present invention provides a protein conjugate comprising a physiologically active polypeptide, non-peptidic polymer and immunoglobulin, which are covalently interlinked to one another.